Serotonin (also referred to as 5-hydroxytryptamine or 5-HT) is a neurotransmitter that has been strongly implicated in the pathophysiology and treatment of a wide variety of neuropsychiatric disorders. Serotonin exerts its effects through a diverse family of serotonin receptor molecules (referred to herein as “5-HT receptors” or “5-HTRs”). Classically, members of the serotonin receptor family have been grouped into seven (7) subtypes pharmacologically, i.e., according to their specificity of various serotonin antagonists. Thus, while all the 5-HT receptors specifically bind with serotonin, they are pharmacologically distinct and are encoded by separate genes. To date, fourteen (14) mammalian serotonin receptors have been identified and sequenced. More particularly, these fourteen separate 5-HT receptors have been grouped into seven (7) pharmacological subtypes, designated 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. Several of the subtypes are further subdivided such that the receptors are grouped pharmacologically as follows: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5-HT3B, 5-HT4, 5-HT5A, 5-HT6, 5HT7. However, when the nucleic and amino acid sequences of the receptors are compared, the percent identity among the subtypes is not correlated to the pharmacological groupings.
Of the fourteen different mammalian serotonin receptors that have been cloned, all but one are members of the G-protein coupled receptor superfamily; that is, they are generally coupled to different second messenger pathways linked through guanine-nucleotide regulatory (G) proteins. For instance, serotonin receptors 5-HT1A, 5-HT1B, and 5-HT1D, inhibit adenylate cyclase, and 5-HT2 receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides. The 5-HT2 receptor belongs to the family of rhodopsin-like signal transducers that are distinguished by their seven-transmembrane configuration and their functional linkage to G-proteins.
The subtypes of serotonin receptors have been historically distinguished on the basis of their pharmacological binding profiles, on second messenger coupling, and based on physiological roles known for the better characterized serotonin receptors. Most of the data in the field used to characterize 5-HT receptors is not based on the properties of a single purified receptor protein or gene, but rather based on experimental observations using a model tissue.
As stated previously elsewhere herein, fourteen separate serotonin receptors have been identified encompassing seven subtypes based on, inter alia, structural homology, second messenger system activation, and drug affinity for certain ligands. Molecular cloning has indicated that 5-HT receptors belong to at least two protein superfamilies: G-protein-associated receptors that have seven putative transmembrane domains (TMDs) (5-HT1A, 1B, 1D, 1E, 5-HT2) and ligand-gated ion channel receptors that have four putative TMDs (5-HT3). The 5-HT2 subfamily is further divided into three classes: 5-HT2A, 5-HT2B, and 5-HT2C. 5HT2A and 5-HT2C receptor antagonists are thought to be useful in treating depression, anxiety, psychosis, and eating disorders. 5-HT2A and 5-HT2C receptors share about 51% amino acid homology overall and approximately 80% homology in the transmembrane domains. Studies of the 5-HT2A receptor in recombinant mammalian cell lines revealed that the receptor possessed two affinity states, high and low.
Both the 5-HT2A and 5-HT2C receptors are coupled to phospholipase C and mediate responses through the phosphatidylinositol pathway. Studies with agonists and antagonists display a wide range of receptor responses suggesting that there is a wide diversity of regulatory mechanisms governing receptor activity. The 5-HT2A and 5-HT2C receptors have also been implicated as the site of action of hallucinogenic drugs.
In the central nervous system (CNS), serotonin is thought to be involved in learning and memory, sleep, thermoregulation, motor activity, pain, sexual and aggressive behaviors, appetite, neuroendocrine regulation, and biological rhythms. Serotonin has also been linked to pathophysiological conditions such as anxiety, depression, obsessive-compulsive disorders, schizophrenia, suicide, autism, migraine, emesis, alcoholism and neurodegenerative disorders.
Serotonin regulates a wide variety of sensory, motor and behavioral functions in the mammalian CNS. This biogenic amine neurotransmitter is synthesized by neurons of the brain stem that project throughout the CNS, with highest density in basal ganglia and limbic structures (Steinbusch, 1984, In: Handbook of Chemical Neuroanatomy 3:68-125, Bjorklund et al., Eds., Elsevier Science Publishers, B.V.). Serotonergic transmission is thought to be involved with a variety of behaviors and psychiatric disorders including anxiety, sleep regulation, aggression, feeding and depression (Cowen, 1991, British J. Psych., 159:7-14; and Lucki, 1992, Neurosci. & Biobehav. Rev., 16:83-93). Understanding how 5-HT mediates its diverse physiological actions requires the identification and isolation of the pertinent 5-HT receptors.
Recently, studies have suggested that serotonin may play a role in the immune system since data demonstrate that serotonin receptors are present on various cells of the immune system. The “mind/body” problem has fascinated people of disparate disciplines for centuries. It has always been understood that there is a link between severe emotions or stress and the immune system. Serotonin is a widely disseminated neurotransmitter and known to play a major role in mood disorders and depression. Its role in modulating the immune response, however, has not been appreciated, much less understood.
It has long been known that the survival of a fetus in utero is an immunological paradox. The fetus, in theory, should undergo allograft rejection by the mother. In most cases, the fetus in not rejected, thus the paradox. Understanding how the maternal immune system selectively suppresses the allograft rejection with regard to the fetus while leaving all of the other immune responses intact has been “the holy grail” of immunology. If one understood the process and could reproduce it therapeutically, it would open a fundamentally new door for potential treatments for autoimmune diseases as well as remarkable new methods for treating the rejection symptoms that accompany transplantation procedures. However, until the present invention, the need for improved therapeutics for autoimmune disease and allograft rejection has been unmet. The present invention meets these needs.
In 1998, Munn et al. (1998, Science 281:1191-1193) solved a major piece of the puzzle. This research group showed that the “rapid T cell-induced rejection of all allogeneic concepti occurred when pregnant mice were treated with a pharmracologic inhibitor of indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme expressed by trophoblasts and macrophages. Thus, by catabolizing tryptophan, the mammalian conceptus suppresses T cell activity and defends itself against rejection.” In other words, shortly after a female becomes pregnant she produces an enzyme (IDO) that sends tryptophan on the first step of the metabolic pathway towards the production of niacin. This obviously implies that tryptophan must, somehow, play a key role in mounting and maintaining an immune response and/or that producing kynurenine (the first step of the niacin pathway) has a suppressive effect. Although it has become clear that the induction of IDO will inhibit T cell proliferation and may play a role in allograft acceptance (Alberati-Giani et al., 1998, Amino Acids 14:251-255; Munn et al., 1999, J. Exp. Med. 189:1362-1373; Widner et al., 2000, Immunol. Today 20:469-473; Pan et al., 2000, Transpl. Immunol. 8:189-194; Mellor et al., 2001, Nature Immunol. 2:64-68), it was absolutely unclear why tryptophan catabolism inhibits the immune responses.
Tryptophan is one of the ten essential amino acids required for building new proteins in the cell. It is possible, though not likely, that the catabolism of tryptophan results in starvation and, therefore, accounts for the observed T cell inhibition. However, none of the other nine essential amino acids have been implicated in the control of T cell responses. Nevertheless, there is a strong correlation between the local depletion of tryptophan levels and inhibition of T cell function (Munn et al., 1999, J. Exp. Med. 189:1362-1373; Widner et al., 2000, Immunol. Today 20:469-473; Frumento et al., 2001, Transplant. Proc. 33:428-430).
Tryptophan is the only known source for producing 5-hydroxytryptamine (also known as serotonin). If the modulation of local tryptophan levels were to be related to the observed modulation in T cell reactivity via the serotonergic pathway, then, obviously, serotonin must play a central role in T cell activation. However, although serotonin is one of the most widely studied biologically active molecules in the history of biochemistry, its role in the T cell activation pathway has not been identified or exploited until the present invention.
There have been reports in the literature about the immunomodulatory effects of adding serotonin exogenously to mitogenically stimulated lymphocyte cultures. Under some circumstances, serotonin has been shown to stimulate the activated T cells (Foon et al., 1976, J. Immunol. 117:1545-1552; Kut et al., 1992, Immunopharmacol. Immunotoxicol. 14:783-796; Young et al., 1993, Immunology 80:395-400), whereas most laboratories report that high concentrations of added serotonin inhibit the proliferation (Slauson et al., 1984, Cell. Immunol. 84:240-252; Khan et al., 1986, Int. Arch. Allergy Appl. Immunol. 81:378-380; Mossner & Lesch, 1998, Brain, Behavior, and Immunity 12:249-271). Thus, the prior art is, at best, unclear as to what role, if any, serotonin might play in modulating the immune response.
Over the intervening years, it has been shown that of the fourteen known pharmacologically distinct serotonin receptors, lymphocytes express type 2a, type 2b, type 2c, type 6 and type 7 on resting cells (Ameisen et al., 1989, J. Immunol. 142:3171-3179; Stefulj et al., 2000, Brain, Behavior, and Immunity 14:219-224) and that the type 1a and type 3 receptors are up-regulated upon activation (Aune et al., 1993, J. Immunol. 151:1175-1183; Meyniel et al., 1997, Immunol. Lett. 55:151-160; Stefulj et al., 2000, Brain, Behavior, and Immunity 14:219-224). Although the functional role of these receptors on lymphocytes has never been clearly defined, it is generally known that the serotonin receptors, except for the type 3 receptors which are cation channels, are 7 transmembrane domain, G-coupled receptors (for a review see Barnes and Sharp, 1999, NeuroPharm. 38:1083-1152). More specifically, the type 1 receptors act on adenylate cyclase, resulting in a down-regulation of cAMP (De Vivo & Maayani, 1986, J. Pharmacol. Exp. Ther. 238:248-252). Forskolin, for example, is a pharmacological agonist of adenylate cyclase and an up-regulator of cAMP, and, therefore, an inhibitor of T cell activation. Forskolin inhibition of T cells, on-the-other-hand, can be rescued by the addition of serotonin (Aune et al., 1990, J. Immunol. 145:1826-1831; Aune et al., 1993, J. Immunol. 151:1175-1183).
In contrast to the type 1a receptors, the type 6 and type 7 receptors, present on resting T cells, act by up-regulating cAMP in response to serotonin (Ruat et al., 1993, Biochem. Biophys. Res. Commun. 193:268-276; Ruat et al., 1993, Proc. Natl. Acad. Sci. USA 90:8547-8551). It is almost a counterintuitive arrangement, the type 6 and 7 receptors present on the resting cells should act to slow the T cell response, whereas the type 1a should counteract the signals sent from the 6 and 7 receptors. The type 2a and 2c receptors couple positively to phospholipase C and lead to increased accumulation of inositol phosphates and intracellular Ca2+, thereby turning on the Protein Kinase C signal transduction cascade (for a review see Boess and Martin, 1994, Neuropharmacology 33:275-317).
With regard to the functional control of the immune response, Gershon et al. (1975, J. Exp. Med. 142:732-738), hypothesized that serotonin was required for mounting a T cell-mediated delayed-type hypersensitivity (DTH) response in mice. However, the authors of this study attributed the dependence of the DTH response on serotonin to the vasoactive properties of this biogenic amine.
A series of studies from the Miles Research Center in West Haven, Conn., showed the presence and involvement of the 5-HT 1a receptors in human and murine T cells (Aune et al., 1990, J. Immunol. 145:1826-1831; Aune et al., 1993, J. Immunol. 151:1175-1183; Aune et al., 1994, J. Immunol. 153:1826-1831). These studies established that IL-2-stimulated human T cell proliferation could be inhibited by a blockade of tryptophan hydroxylase, i.e., the first enzyme involved in the conversion of tryptophan to serotonin, and that the inhibition could be reversed by the addition of 5-hydroxy tryptophan, i.e., the metabolic product of the inhibited enzyme. Furthermore, they could block human T cell proliferation in vitro with a 5-HT 1a-specific receptor antagonist. In a murine model, they demonstrated that a type 1a receptor antagonist, but not a type 2 receptor antagonist, was able to inhibit the in vivo contact sensitivity response, but not antibody responses, to oxazalone.
Using both type 1a and type 2 receptor antagonist, Laberge et al. (1996, J. Immunol. 156:310-315) serotonin could induce the chemotactic factor, IL-16, from CD8+ T cells and that this activity could be specifically inhibited by the addition of type 2 receptor inhibitors, but not antagonists of the 1a receptor. Thus, although the prior art indicated that serotonin plays a role in the immune system, it was not clear what that role was and there was nothing to suggest that the immune system could be modulated by use of receptor antagonists.
There are a handful of references suggesting that serotonin may play a role the immune response. In 1989, a prominent immunologist, Philip Askenase, and his colleagues demonstrated that a 5-HTR2 antagonist could inhibit a delayed-type hypersensitivity (DTH) response in mice (Amiesen et al., 1989, J. Immunol. 142:3171 -3179). Amiesen et al., reasoned that “late-acting DTH effector T cells might express functional 5-HT2R and that these receptors might require in vivo activation in order for the T cells to locally produce the inflammatory lymphokine-dependent aspects of DTH.” These data were subsequently orphaned presumably because rodent mast cells contain serotonin but human mast cells do not, such that the results were not applicable to a human immune response. Later, Aune et al. (1994, J. Immunol. 153:489-498), demonstrated that a 5-HTR1a antagonist could inhibit a murine DTH response in vivo and showed that inhibition of the enzyme tryptophan hydroxylase (the first enzyme involved in the conversion of tryptophan to serotonin) could inhibit T cell proliferation. Again, these authors provided important pieces of information, but failed to recognize the larger role of serotonin in the mounting of a T cell-dependent response.
The first evidence that macrophages and lymphocytes expressed receptors capable of responding to serotonin was presented in 1984 (Roszman et al., 1984, Soc. Neurosci. 10:726). Over the intervening years, it has been shown that of the fourteen known pharmacologically distinct serotonin receptors, resting lymphocytes express 5-HT2A, 2B, 2C, 6, and 7 (Ameisen et al., 1989, J. Immunol. 142:3171-3179; Stefulj et al., 2000, Brain, Behavior, and Immunity 14:219-224) and that the 5-HT1A and 5-HT3 receptors are up-regulated upon activation (Aune et al., 1993, J. Immunol. 151:1175-1183; Meyniel et al., 1997, Immunol. Lett. 55:151-160; Stefulj et al., 2000, Brain, Behavior, and Immunity 14:219-224).
Although the functional role of serotonin receptors on lymphocytes and in immune regulation if any has never been defined, it is generally known that serotonin receptors, with the exception of type 3 receptors which are cation channels, are G-coupled receptors comprising seven transmembrane domains (for a review see Barnes and Sharp, 1999, NeuroPharm. 38:1083-1152). More specifically, the type 1 receptors act on adenylate cyclase, resulting in a down-regulation of cAMP (De Vivo & Maayani, 1986, J. Pharmacol. Exp. Ther. 238:248-252).
In contrast to the 5-HT1A receptors, the 5-HT6 and 5-HT7 receptors, present on resting T cells, act by up-regulating cAMP in response to serotonin (Ruat et al., 1993, Biochem. Biophys. Res. Commun. 193:268-276; Ruat et al., 1993, Proc. Natl. Acad. Sci. USA 90:8547-8551). In an apparently counterintuitive arrangement, the 5-HT6 and 5-HT7 receptors present on the resting cells should act to slow the T cell response, whereas the type 1a should counteract the signals sent from the 5-HT6 and 5-HT7 receptors. The 5-HT2A and 5-HT2C receptors couple positively to phospholipase C and lead to increased accumulation of inositol phosphates and intracellular Ca2+, thereby turning on the protein kinase C signal transduction cascade (for a review see Boess and Martin, 1994, Neuropharmacology 33:275-317).
It was previously hypothesized that serotonin was required for mounting a T cell-mediated delayed-type hypersensitivity (DTH) response in mice (Gershon et al., 1975, J. Exp. Med. 142:732-738). It was concluded that dependence of the DTH response on serotonin was due to the vasoactive properties of this biogenic amine. There have been mixed reports in the literature about the immunomodulatory effects of serotonin. Under some circumstances, exogenous 5-HT has been shown to stimulate activated T cells (Foon et al., 1976, J. Immunol. 117:1545-1552; Kut et al., 1992, Immunopharmacol. Immunotoxicol. 14:783-796; Young et al., 1993, Immunology 80:395-400), whereas most laboratories report that high concentrations of exogenous 5-HT inhibit proliferation of activated T cells (Slauson et al., 1984, Cell. Immunol. 84:240-252; Khan et al., 1986, Int. Arch. Allergy Appl. Immunol. 81:378-380; Mossner & Lesch, 1998, Brain, Behavior, and Immunity 12:249-271). Thus, it is not clear what effect if any serotonin may have on the immune system, since studies suggest that this neurotransmitter both up- and down-regulates the immune response.
There exists a long-felt need to develop therapies for modulating the immune response, especially therapies that regulate certain aspects of the immune response while not affecting others. Thus, there is a great need to identify potential therapeutic targets for modulating the immune response. The present invention meets these needs.